Compositions and methods for preventing or treating inflammatory bowel disease

ABSTRACT

Compositions comprising docosahexaenoic acid (DHA) and optionally one or more fatty acids selected from the group consisting of eicosapentaenoic acid (EPA), arachidonic acid (ARA), linoleic acid (LA), and α-linoleic acid (ALA) are administered to felines for preventing or treating feline inflammatory bowel disease (IBD).

CROSS-REFERENCE TO PRIOR APPLICATIONS

This application claims priority from U.S. Provisional Application No.60/754,806 filed on Dec. 29, 2005.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to compositions and methods forpreventing or treating inflammatory bowel disease and particularly tothe use of docosahexaenoic acid for preventing or treating felineinflammatory bowel disease.

2. Description of the Related Art

The terms “inflammatory, bowel disease” or “IBD” refer to a group ofchronic idiopathic gastrointestinal disorders characterized byinflammatory infiltrates within the lamina propria of thegastrointestinal tract. IBD encompasses segmental granulomatousenterocolitis, lymphoplasmacytic enteritis, eosinophilicgastroenterocolitis, lymphocytic gastroenterocolitis, suppurativeenterocolitis, and histiocytic colitis. The lymphoplasmacytic form isprobably the most common type of IBD. These specific types of IBD arecharacterized based on the type of inflammatory infiltrate found in thelamina propria. The inflammatory infiltrates can be quite variable interms of severity and cell types, with lymphocytes and plasma cellsbeing the most common cell types. Inflammatory infiltrates may involvethe stomach, small bowel, and colon. In cats, for example, the stomachand small bowel are affected most often. In many cases, multiplesegments of the bowel are involved and clinical signs may be mixed,reflecting the broad distribution of mucosal lesions. The severity ofIBD varies from mild clinical signs to life-threatening protein-losingenteropathies.

Mucosal inflammatory infiltrates are responsible for the clinicalmanifestations of IBD. Mucosal inflammation disrupts normal absorptiveprocesses. Such disruption results in malabsorption and osmoticdiarrhea. Altered gut permeability can result in leakage of fluid,protein, and blood into the gut lumen. Malabsorbed fats, carbohydrates,and bile acids result in secretory diarrhea. Inflammatory mediators mayalso directly trigger intestinal secretion and mucus production bygoblet cells. Mucosal inflammatory infiltrates may alter intestinal andcolonic motility patters, a mechanism attributed to the influence ofprostaglandins and leukotrienes on smooth muscle. Inflammation of thestomach and small bowel stimulates receptors that trigger vomiting. Incats, for example, the most common clinical signs of IBD are chronicvomiting, diarrhea, and weight loss.

The fundamental pathway for the development of IBD involveshypersensitivity. Two related theories attempt to explain the underlyingcause for hypersensitivity reactions. The first theory speculates thatfelines with IBD develop a defect in the intestinal mucosal barrier.Loss of mucosal integrity results in increased gut permeability andhypersensitivity responses to allergens that are normally tolerated. Thesecond theory speculates that IBD results from aberrant immunologicalresponses to luminal antigens. Both potential pathways culminate inrelease of inflammatory mediators. These substances may further damagethe intestinal mucosal surface and set up a cycle of inflammation andloss of barrier function.

Essential fatty acids have specific roles in cell function regulation.For example, the omega-3 eicosapentaenoic acid (EPA), and the omega-6arachidonic and gamma-linolenic acids are precursors for the synthesisof eicosanoids which are immunoregulatory molecules functioning as localhormones and mediators of inflammation. The eicosanoids synthesized fromarachidonic acid (ARA) are proinflammatory compared to eicosanoidsproduced from eicosapentaenoic and gamma-linolenic acids, and may resultin pathologic conditions when produced in excessive amounts. Macrophagesare a significant source of eicosanoids, and modulate the intensity andduration of inflammatory and immune responses. The predominantpolyunsaturated fatty acid in membrane phospholipids of macrophages andlymphocytes is ARA. Administration of gamma-linolenic or EPA results inthe replacement of ARA in the macrophage membrane with eicosapentaenoicor gamma-linolenic acid. The result of such replacement is theproduction of fewer ARA-derived eicosanoids and more EPA-derived orgamma-linolenic acid-derived eicosanoids, thereby reducing theimmunologic response to an inflammatory episode.

A definitive diagnosis of IBD is based on the histopathologicalexamination of mucosal or full-thickness intestinal biopsy specimenscollected by endoscopic or surgical techniques. Thus, there is a needfor alternative methods for diagnosing feline IBD that are less invasivethan obtaining biopsy specimens. There is also a need for new methodsand compositions useful for preventing and treating feline IBD.

SUMMARY OF THE INVENTION

It is, therefore, an object of the invention to provide methods forpreventing or treating IBD in felines.

It is another object of the invention to provide compositions suitablefor preventing or treating IBD in felines.

It is another object of the invention to provide methods for determiningif a feline is suffering from IBD.

It is a further object of the invention to provide articles ofmanufacture in the form of kits that contain combinations of foods,compounds, ingredients, and devices useful for preventing or treatingIBD in felines.

It is another object of the invention to provide means for communicatinginformation about the methods, compositions, articles of manufacture,and benefits of the invention.

One or more of these and other objects are achieved using novel methodsfor preventing and/or treating IBD in felines susceptible to orsuffering from IBD comprising administering to the felines atherapeutically-effective amount of docosahexaenoic acid (DHA). Methodsfor diagnosing IBD and kits comprising combinations of foods, compounds,ingredients, and devices useful for preventing and/or treating IBD arealso provided.

Additional objects, features, and advantages of the invention will beapparent to those skilled in the art.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention provides methods for treating IBD in afeline suffering from IBD. Treating IBD includes ameliorating,suppressing, and/or eradicating IBD. Those skilled in the art candiagnose IBD (and distinguish IBD from other gastrointestinal diseases)utilizing diagnostic tests (e.g. complete blood cell count, serumbiochemistry, serum thyroxine level, immunodeficiency virus test, felineleukemia virus test, urinalysis, fecal examinations for parasites andbacteria); dietary trials; abdominal radiographs and/or ultrasound;and/or examination of mucosal or full-thickness intestinal biopsyspecimens. In another aspect, the invention provides methods forpreventing IBD in a feline susceptible to developing IBD. Preventing IBDincludes reducing the risk of IBD, delaying the onset of IBD, and/orkeeping a feline from developing IBD. The methods comprise administeringto the feline a therapeutically-effective amount of docosahexaenoic acid(DHA). A therapeutically-effective amount is an amount that will achievethe goal of treating IBD when the feline is suffering from IBD, orpreventing IBD when the feline is susceptible to developing IBD, orlowering the level of CD4+ lymphocytes and neutrophils in a felinesusceptible to or suffering from IBD. Administering means introducingDHA or other compounds into the feline in a suitable dosage form by asuitable administration route (e.g. orally, topically, or parenterally).DHA can be administered, for example, as pure DHA or DHA derivative(e.g., a salt such as an ester) or as a composition comprising DHAand/or DHA derivative(s). References herein to DHA and other fatty acidsherein include the derivatives of such compounds. A DHA-comprisingcomposition may also comprise one or more conventionalpharmaceutically-acceptable excipients (e.g., adjuvants, carriers,and/or vehicles). In some embodiments, the DHA-comprising compositionmay comprise a food composition.

The invention is based upon the surprising discovery that DHA, but notEPA or other similar fatty acids, is useful for preventing or treatingIBD in felines and that DHA may have the opposite effect in otheranimals. While EPA and related fatty acids alone or in combination donot prevent or treat IBD, they are useful for supplementing DHA inpreventing or treating IBD. Thus, the unexpected result that DHA aloneor DHA in combination with EPA and related fatty acids is effective forpreventing or treating IBD when administered to felines in atherapeutically-effective amount.

DHA effectively lowers the level of CD4+ lymphocytes and neutrophils ina feline, including a feline susceptible to or suffering from IBD, whenDHA is administered in a therapeutically-effective amount, as describedherein.

In some embodiments, the methods comprise administering to the felinefrom about 6 to about 165 mg/kg body weight/day DHA. In some suchembodiments, from about 12 to about 65 mg/kg body weight/day DHA isadministered to the feline. In others, from about 12 to about 32 mg/kgbody weight/day DHA is administered to the feline. The daily amount ofDHA can be administering in a single dose or, alternatively, in two ormore dosages that make up the daily dose.

In some embodiments, administering DHA comprises feeding DHA to thefeline, i.e., feeding DHA or a composition comprising DHA (including DHAderivatives).

In various embodiments, a DHA-comprising composition fed to the felinecomprises a food composition. In some embodiments, the food compositionmeets the AAFCO's minimum nutrient level requirements for reproductionor maintenance. See 2005 AAFCO Official Publication, pages 137-40. Insome embodiments, the food composition comprises a dry food. In others,the food composition comprises a semi-moist food. In still others, thefood composition comprises a moist food. The terms, “dry”, “moist” and“semi-moist”, as used herein, are familiar to one of skill in the art.The food composition may be a supplement, treat snack, or partially orfully edible toy. In some embodiments, the composition comprises amixture of one or more foods or a hypoallergenic food composition.

In some embodiments, the feline is a companion feline. A companionfeline can be a feline kept as a pet. A companion feline can also be afeline from a widely domesticated species, for example, cats (Felisdomesticus) regardless of whether or not it is kept as a pet. In someembodiments, the feline is a growing feline. A growing feline is onethat has not reached adult size. For example, a growing cat typically isone that is less than about one year old. In some embodiments, thefeline is an adult feline. An adult feline is a feline of any age afterjuvenile growth and development has been completed, including senior andgeriatric felines. For example, an adult cat typically is one that isfrom about one year old through the remainder of its life. A seniorfeline is one of an age at which it is at a risk for suffering from anage-related disease regardless of whether or not the feline showsobvious physical or behavioral signs of aging. For example, a senior cattypically is a cat from about seven to about eleven years old. Ageriatric feline is a feline showing signs of advanced age. For example,a geriatric cat typically is a cat of about twelve years of age andbeyond.

Unless otherwise stated, all percentages herein are weight percentageson a dry matter basis. The term “dry matter basis” means that aningredient's concentration in a composition is measured after anymoisture in the composition is removed.

In some embodiments, the composition administered to the felinecomprises from about 0.05 to about 1% DHA. In some such embodiments, thecomposition comprises from about 0.1 to about 0.4% DHA. In others, thecomposition comprises from about 0.1 to about 0.2% DHA. In yet othersuch embodiments, the composition comprises from about 0.05 to about0.2% DHA. In further such embodiments, the composition comprises fromabout 0.05 to about 0.15% DHA. And in yet further such embodiments, thecomposition comprises from about 0.05 or about 0.1 to about 0.15, about0.2, or about 0.4% DHA.

In additional embodiments, the composition administered to the felinefurther comprises at least one fatty acid selected from the groupconsisting of eicosapentaenoic acid (EPA), arachidonic acid (ARA),linoleic acid (LA), and α-linoleic acid (ALA). In some such embodiments,the composition comprises from about 0.05 to about 1% of each fatty acidpresent in the composition. In other such embodiments, the compositioncomprises from about 0.1 to about 0.5% or from 0.1 to about 0.3% of thefatty acid. In one embodiment, the composition administered to thefeline further comprises from about 0.05 to about 1% EPA. In some suchembodiments, the composition comprises from about 0.1 to about 0.5% EPA.In other such embodiments, the composition comprises from about 0.1 toabout 0.3% EPA. In yet other such embodiments, the composition comprisesfrom about 0.05 to about 0.3% EPA. In further such embodiments, thecomposition comprises from about 0.15 to about 0.3% EPA. In yet furthersuch embodiments, the composition comprises from about 0.05, about 0.1,or about 0.15 to about 0.2, about 0.3, about 0.4, or about 0.5% EPA.

In some embodiments, the composition administered to the felinecomprises from about 0.05 to about 1% DHA and from about 0.05 to about1% EPA, and the ratio of the amount of EPA present in the composition tothe amount of DHA in the composition is from about 1 to about 2. In somesuch embodiments, the ratio of the amount of EPA present in thecomposition to the amount of DHA present in the composition is fromabout 1.2 to about 1.8. In other such embodiments, the ratio of theamount of EPA in the composition to the amount of DHA in the compositionis from about 1.2 to about 1.5. In yet other such embodiments, the ratioof the amount of EPA present in the composition to the amount of DHApresent in the composition is from about 1.3 to about 1.6. And infurther such embodiments, the ratio of the amount of EPA present in thecomposition to the amount of DHA present in the composition is fromabout 1, about, 1.2, or about 1.3 to about 1.5, about 1.6, about 1.8, orabout 2.

In some embodiments, the methods of prevention and treatment furthercomprise administering to the feline an anti-inflammatory bowel disease(anti-IBD) agent in conjunction with administering DHA or thecombination of DHA and at least one fatty acid selected from the groupconsisting of EPA, ARA, LA, and ALA. An anti-IBD agent is a compound, aderivative thereof (e.g., a salt, solvate, or hydrate of the compound),or a composition comprising such compounds and/or derivatives that isused to prevent or treat IBD. “In conjunction” means that an anti-IBDagent is administered to the feline either together with DHA orseparately from DHA at the same or different frequency via the same ordifferent administration route and either at about the same time as DHAor periodically. “At about at the same time” generally means that theanti-IBD agent is either administered when DHA is administered to thefeline or within about 72 hours after administering DHA to the feline.“Periodically” generally means that an anti-IBD agent is administered toa feline following a dosage schedule suitable for administering theagent while a DHA-comprising composition is fed to the feline routinelyas appropriate for that feline. Thus, the term “in conjunction”specifically includes situations when an anti-IBD agent is administeredto a feline for a prescribed period of time while DHA is administered tothe feline for a much longer period of time (e.g. for life). If morethan one agent is administered to a feline, the dosage form and route ofadministration for each agent may vary. Those skilled in the art wouldunderstand that one or more anti-IBD agents can be administered to afeline while the feline is fed a single DHA-comprising composition or,alternatively, when the feline is fed different DHA-comprisingcompositions for varying time intervals.

Suitable anti-IBD agents include, for example, corticosteroids (e.g.,prednisone, prednisolone), immunosuppresants (e.g. azathiprine), andantibiotics (e.g., metronidazole, amoxicillin, tylosin). Anti-IBD agentscan be administered, for example, in the form of salts derived frominorganic or organic acids. Depending on the particular compound, a saltof the compound may be advantageous due to one or more of the salt'sphysical properties, for example, enhanced pharmaceutical stability indiffering temperatures and humidity, or a desirable solubility in wateror oil. The salt preferably is a pharmaceutically-acceptable salt.

The preferred total daily dose of an anti-IBD agent (administered ineither single or divided doses) is typically from about 0.001 to about100 mg/kg body weight, more preferably from about 0.01 to about 30 mg/kgbody weight, and even more preferably from about 0.01 to about 10 mg/kgbody weight. Dosage unit compositions can contain such amounts andsubmultiples thereof to make up the daily dose. In many instances, theadministration of the anti-IBD agent will be repeated a plurality oftimes. Multiple doses per day typically may be used to increase thetotal daily dose, if desired. Factors affecting the preferred dosageregimen include, for example, the age, weight, and condition of thefeline; the severity of the disease; the route of administration;pharmacological considerations, such as the activity, efficacy,pharmacokinetic, and toxicology profiles of the particular anti-IBDagent used; whether a drug delivery system is utilized; and whether theanti-IBD agent is administered as part of a drug combination. Thus, thedosage regimen can vary widely, and therefore, can differ from thepreferred dosage regimen discussed above.

In yet another aspect, the invention provides compositions suitable forpreventing and/or treating IBD in a feline. These compositions aredescribed and exemplified in the context of the methods herein forpreventing and/or treating IBD in a feline.

In a further aspect, the invention provides methods for preparing thecompositions suitable for use in methods of prevention and treatment ofIBD. Such compositions can be prepared, for example, by mixing two ormore ingredients (including food compositions) that, when combined,yield a composition of the invention or by mixing one or more foodcompositions with additional ingredient(s) such as, for example, DHA,EPA, and/or an anti-IBD agent. Such compositions can also be prepared byone or more of the methods discussed in, for example, Small AnimalNutrition, pages 127-46 (2000).

In yet further aspect, the invention provides for a use of DHA andoptionally at least one fatty acid selected from the group consisting ofEPA, ARA, LA, and ALA to prepare a composition of the invention suitablefor preventing or treating feline IBD. In some embodiments, theinvention provides a use of DHA to prepare a composition comprising fromabout 0.05 to about 1% DHA. In some such embodiments, the compositioncomprises from about 0.1 to about 0.4% DHA. In others, the compositioncomprises from about 0.1 to about 0.2% DHA. In yet other suchembodiments, the composition comprises from about 0.05 to about 0.2%DHA. In further such embodiments, the composition comprises from about0.05 to about 0.15% DHA. In yet further such embodiments, thecomposition comprises from about 0.05 or about 0.1 to about 0.15, about0.2, or about 0.4% DHA. In some embodiments, the composition comprises afood composition.

In some embodiments, the invention provides a use of DHA and at leastone fatty acid selected from the group consisting of EPA, ARA, LA, andALA, preferably EPA, to prepare a composition comprising from about 0.05to about 1% DHA and from about 0.05 to about 1% of each of EPA, ARA, LA,and/or ALA present in the composition to prevent and/or treat felineIBD. In some such embodiments, the composition comprises from about 0.1to about 0.4% DHA and from about 0.1 to about 0.5% of one or more ofEPA, ARA, LA, and/or ALA. In other such embodiments, the compositioncomprises from about 0.1 to about 0.2% DHA and from about 0.1 to about0.3% of one or more of EPA, ARA, LA, and/or ALA. In yet other suchembodiments, the composition comprises from about 0.05 to about 0.2% DHAand from about 0.05 to about 0.3% of one or more of EPA, ARA, LA, and/orALA. In further such embodiments, the composition comprises from about0.05 to about 0.15% DHA and from about 0.15 to about 0.3% of one or moreof the fatty acids. In yet further such embodiments, the compositioncomprises from about 0.05 or about 0.1 to about 0.15, about 0.2, orabout 0.4% DHA and from about 0.05, about 0.1, or about 0.15 to about0.2, about 0.3, about 0.4, or about 0.5% of one or more of the fattyacids. In some embodiments, the composition comprises a foodcomposition.

In some embodiments, the invention provides a use of DHA and EPA toprepare a composition comprising from about 0.05 to about 1% DHA andfrom about 0.05 to about 1% EPA wherein the ratio of the amount of EPApresent in the composition to the amount of DHA present in thecomposition is from about 1 to about 2 to prevent and/or treat felineIBD. In some such embodiments, the ratio of the amount of EPA in thecomposition to the amount of DHA present in the composition is fromabout 1.2 to about 1.8. In other such embodiments, the ratio of theamount of EPA present in the composition to the amount of DHA present inthe composition is from about 1.2 to about 1.5. In other suchembodiments, the ratio of the amount of EPA present in the compositionto the amount of DHA present in the composition is from about 1.3 toabout 1.6. In further such embodiments, the ratio of the amount of EPApresent in the composition to the amount of DHA in the composition isfrom about 1, about, 1.2, or about 1.3 to about 1.5, about 1.6, about1.8, or about 2. In some embodiments, the composition comprises a foodcomposition.

In a further aspect, the invention provides a method for determining ifa feline is suffering from IBD. The method comprises dividinglymphocytes collected from blood obtained from the feline into a first,second, and third sample comprising equal amounts of lymphocytes;exposing the second sample to an amount of a mitogen for a period oftime; exposing the third sample to the same amount of the same mitogenas the second sample for the same period of time as the second sample inthe presence of an amount of DHA; and comparing the levels of lymphocyteproliferation in all samples. The feline has IBD if the level oflymphocyte proliferation in the second sample is higher than the levelof lymphocyte proliferation in the first sample, and the level oflymphocyte proliferation in the third sample is lower than the level oflymphocyte proliferation in the second sample.

Lymphocytes are collected from blood obtained from a feline and aredivided into samples comprising equal amounts of lymphocytes. Proceduresfor obtaining blood from felines, isolating the lymphocytes from thatblood, and counting the lymphocytes are known to those skilled in theart. In some embodiments, lymphocytes can be collected as described inExample 1. The lymphocytes isolated from a feline's blood are dividedinto three or more samples with all samples comprising equal amounts oflymphocytes. In some embodiments, the lymphocytes are divided into threesamples (i.e., a first, second, and third sample). One of those samples(i.e., the first sample) is used as a control. One of the remaining twosamples (i.e., the second sample) is exposed to an amount of mitogen fora period of time, and the other (i.e., the third sample) is exposed tothe same amount of mitogen as the second sample for the same period oftime as the second sample in the presence of an amount of DHA. Allsamples are incubated for the same period of time at the sametemperature.

A mitogen is an agent that triggers mitosis. Any mitogen that cantrigger mitosis of feline lymphocytes is suitable for the method of theinvention. In some embodiments, the mitogen is a polyclonal mitogen. Apolyclonal mitogen is a mitogen that induces mitosis in lymphocytes ofmany different specificities or clonal origins. Mitogens suitable forthe method of IBD diagnosis of the invention include, for example,phytohemagglutinin (PHA), concanavalin (ConA), pokeweed mitogen (PWM),lipopolysaccharide (LPS), and anti-CD3 antibody.

Procedures for measuring lymphocyte proliferation are known to thoseskilled in the art. Any procedure for measuring in vitro lymphocyteproliferation is suitable for the method of the invention. In vitrolymphocyte proliferation can be measured directly (e.g., by countingcells or by determining the mitotic index) or indirectly (e.g. bydetermining the rate of overall metabolic activity in a lymphocytepopulation or by monitoring the synthesis of deoxyribonucleic acid(DNA)). In some embodiments, in vitro lymphocyte proliferation ismeasured as discussed in Example 1.

In some embodiments of the method of the invention, lymphocyteproliferation is measured by monitoring DNA synthesis. Procedures formonitoring and measuring DNA synthesis are known to those skilled in theart. Any procedure for monitoring and measuring DNA synthesis issuitable for the method of the invention. DNA synthesis can be monitoredand measured by, for example, labeling the DNA of mitotically activecells with ³H-thymidine or 5-bromo-2′-deoxyuridine (BrdU) and thendetermining the amount of ³H-thymidine or BrdU that was incorporatedinto DNA. In some embodiments, DNA synthesis is measured as discussed inExample 1.

As discussed above, a determination if a feline is suffering from IBD ismade by comparing the levels of in vitro lymphocyte proliferation in thefirst, second and third samples. If the level of lymphocyteproliferation in the second sample (i.e., the sample treated with amitogen, but no DHA) is higher than the level of lymphocyteproliferation in the first sample (i.e., the control sample that was nottreated with mitogen or DHA), then the mitogen used in the assay hasindeed stimulated in vitro lymphocyte proliferation in the second andthird sample. If that is the case, then the level of lymphocyteproliferation in the third sample (i.e., the sample treated with boththe mitogen and DHA) is compared to the level of lymphocyteproliferation in the second sample. If the level of lymphocyteproliferation in the third sample is lower than the level of lymphocyteproliferation in the second sample, then DHA had inhibited thepro-inflammatory effect of the mitogen indicating that the feline hasIBD.

In a further aspect, the invention provides an article of manufacture inthe form of a kit suitable for preventing or treating IBD. The kitcomprises DHA or a DHA-comprising composition of the invention. In someembodiments, the kit further comprises an anti-IBD agent (i.e., the kitcomprises one or more anti-IBD agents). In some embodiments, the kitfurther comprises instructions for one or more of (a) administering DHAto a feline, (b) administering an anti-IBD agent to a feline inconjunction with administering DHA to the feline, (c) preventing and/ortreating IBD in a feline by administering DHA to the feline, and (d)preventing and/or treating IBD in a feline by administering an anti-IBDagent in conjunction with administering DHA to the feline.

In some embodiments, the kit comprises a DHA-comprising foodcomposition. In some embodiments, the kit further comprises an anti-IBDagent. In some embodiments, the kit further comprises instructions forone or more of (a) feeding the DHA-comprising food composition to afeline, (b) administering an anti-IBD agent to a feline in conjunctionwith feeding the DHA-comprising food composition to the feline, (c)preventing and/or treating IBD in a feline by feeding the feline aDHA-comprising food composition, and (d) preventing and/or treating IBDin a feline by administering to the feline an anti-IBD agent inconjunction with feeding the feline a DHA-comprising food composition.

In a further aspect, the invention provides an article of manufacture inthe form of a kit comprising two or more ingredients that, when combinedtogether and, optionally, with additional ingredients that are or arenot a part of the kit, yield a DHA-comprising composition of theinvention suitable for preventing and/or treating IBD in a feline. Oneof the two or more ingredients that are to be combined can be, forexample, pure DHA or derivative thereof or a composition comprising DHA.Another one of the two or more ingredients that are to be combined canbe, for example, a food composition. If, to prepare a composition,additional ingredients that are or are not a part of the kit are needed,the kit provides instructions about those ingredients. In someembodiments, the kit further comprises an anti-IBD agent. In someembodiments, the kit further comprises instructions for one or more of(a) preparing the composition by combining the two or more ingredientsand, optionally, additional ingredients that are or are not a part ofthe kit, (b) feeding the composition to a feline to, for example,prevent and/or treat IBD, (c) administering an anti-IBD agent to thefeline in conjunction with feeding the feline the composition, (d)preventing and/or treating IBD in a feline by feeding the feline thecomposition, and (e) preventing and/or treating IBD in a feline byadministering to the feline an anti-IBD agent in conjunction withfeeding the feline the composition.

In some embodiments, the kit comprises in separate containers in asingle package or in separate containers in a virtual package, asappropriate for the kit component, either (A) DHA, (B) a compositioncomprising DHA, or (C) two or more ingredients that, when combinedtogether, and, optionally, with additional ingredients that are or arenot a part of the kit, yield a composition comprising DHA, and one ormore of (1) at least one fatty acid selected from the group consistingof EPA, ARA, LA, and ALA, (2) a food composition suitable forconsumption by a feline susceptible to or suffering from inflammatorybowel disease, (3) an anti-IBD agent, and (4) instructions for one ormore of (a) preparing a composition comprising DHA alone or incombination with at least one fatty acid selected from the groupconsisting of EPA, ARA, LA, and ALA, (b) preparing a food compositionsuitable consumption by a feline susceptible to or suffering frominflammatory bowel disease comprising a therapeutically-effective amountof DHA alone or in combination with at least one fatty acid selectedfrom the group consisting of EPA, ARA, LA, and ALA, (c) administeringDHA alone or in combination with at least one fatty acid selected fromthe group consisting of EPA, ARA, LA, and ALA to a feline to preventand/or treat IBD, (d) preventing and/or treating IBD in a feline byfeeding the feline a composition comprising DHA alone or in combinationwith at least one fatty acid selected from the group consisting of EPA,ARA, LA, and ALA, (e) administering an anti-IBD agent to a feline inconjunction with feeding the feline a composition comprising DHA aloneor in combination with at least one fatty acid selected from the groupconsisting of EPA, ARA, LA, and ALA, and (f) preventing and/or treatingIBD in a feline by administering to the feline an anti-IBD agent inconjunction with feeding the feline a composition comprising DHA aloneor in combination with at least one fatty acid selected from the groupconsisting of EPA, ARA, LA, and ALA.

The term “single package” generally means that the components of a kitare physically associated in or with one or more containers andconsidered as a unit of manufacture, distribution, sale, or use.Containers include, for example, bags, boxes, bottles, shrink wrappackages, stapled or otherwise fixed components, and combinationsthereof. A single package can be, for example, containers or individualfood compositions physically associated such that they are considered aunit for manufacture, distribution, sale, or use. The term “virtualpackage” generally means that the components of a kit are associated bydirections on one or more physical or virtual kit components instructingthe user how to obtain additional components, e.g., in a bag containingone component and directions instructing the user to go to a website,contact a recorded message, view a visual message, or contact acaregiver to obtain instructions on how to use the kit. When the kitcomprises a virtual package, the kit is limited to instructions in avirtual environment with one or more physical kit components.

In a further aspect, the invention provides a means for communicatinginformation about or instructions for one or more of (1) using DHA or acomposition comprising DHA to prevent and/or treat IBD in a feline, (2)preventing and/or treating IBD in a feline by administering to thefeline an anti-IBD agent in conjunction with feeding the feline DHA or acomposition comprising DHA, and (3) using a kit of the invention forpreventing and/or treating IBD in a feline comprising a document,digital storage media, optical storage media, audio presentation, orvisual display containing the information or instructions. In someembodiments, the communicating means comprises a document, digitalstorage media, optical storage media, audio presentation, or visualdisplay containing the information or instructions. Preferably, thecommunication means is a displayed web site or a brochure, productlabel, package insert, advertisement, or visual display containing suchinformation or instructions. Useful information or instructions include,for example, (1) information and instructions how to use a composition,method, or kit of the invention and (2) contact information for animalcaregivers if they have a question about the invention and its uses.

In a further aspect, the present invention provides for a use of DHA andoptionally at least one fatty acid selected from the group consisting ofEPA, ARA, LA, and ALA to prepare a medicament. In another, the inventionprovides for the use of a therapeutically-effective amount of such fattyacid(s) to prepare a medicament for preventing or treating feline IBD.Generally, medicaments are prepared by admixing a compound orcomposition with excipients, buffers, binders, plasticizers, colorants,diluents, compressing agents, lubricants, flavorants, moistening agents,and other ingredients known to skilled artisans to be useful forproducing medicaments and formulating medicaments that are suitable foradministration to an animal.

The invention is not limited to the particular methodology, protocols,and reagents described herein because they may vary. Further, theterminology used herein is for the purpose of describing particularembodiments only and is not intended to limit the scope of the presentinvention. As used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural reference unless the context clearlydictates otherwise. Similarly, the words “comprise”, “comprises”, and“comprising” are to be interpreted inclusively rather than exclusively.

Unless defined otherwise, all technical and scientific terms and anyacronyms used herein have the same meanings as commonly understood byone of ordinary skill in the art in the field of the invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice of the present invention, thepreferred methods, devices, and materials are described herein.

All patents, patent applications, and publications mentioned herein areincorporated herein by reference to the extent allowed by law for thepurpose of describing and disclosing the compounds, processes,techniques, procedures, technology, articles, and other compositions andmethods disclosed therein that might be used with the present invention.However, nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

EXAMPLES

The invention can be further illustrated by the following examples,although it will be understood that the examples are included merely forpurposes of illustration and are not intended to limit the scope of theinvention unless otherwise specifically indicated.

Example 1

This example illustrates the effect of DHA and EPA on in vitrolymphocyte proliferation of lymphocytes obtained from the blood ofhealthy cats and cats with IBD.

The study utilizes eleven healthy cats and eleven cats with IBD, Thecats with IBD are diagnosed by an intestinal biopsy, and have a historyof chronic diarrhea. For six weeks, all cats are fed a nutritionallyadequate dry food indicated for gastrointestinal distress. Blood isdrawn from all cats at four, five, and six weeks. Samples with equalamounts of lymphocytes are used in an in vitro lymphocyte proliferationassay.

More specifically, 4.5 ml blood is mixed with 4.5 ml HBSS (Hank'sBalanced Salt Solution)+25 mM HEPES. 4 ml of Ficoll-Paque Plus(Amersham) are slowly injected under the diluted blood. The mixture iscentrifuged for twenty minutes at 500 g at 25° C. The upper layer isdiscarded. The lymphocytes are transferred into a clean tube,resuspended in 6 ml of HBSS+25 mM HEPES, and then centrifuged for tenminutes at 200 g at 25° C. The supernatant is discarded, and thelymphocytes are washed with 6 ml of HBSS. This centrifugation and washstep is repeated two more times. The washed lymphocytes are thenresuspended in 5 ml of AIM medium (Invitrogen). Samples with 200,000lymphocytes in 100 μl of AIM medium are used for the lymphocyteproliferation assay.

The lymphocyte proliferation assay is performed utilizing Amersham'sBiotrak cell proliferation ELISA system, version 2. Lymphocyte samplesare incubated in a 96-well plate with 7 μg/ml PHA (phytohemagglutinin)in the absence or presence of 12.5 μM and 25 μM DHA or EPA in a 37° C.incubator with 5% CO₂ and 90% humidity for forty hours. After that, 20μl of 100 μM BrdU is added, and the samples are incubated for two hoursat 37° C. The plates are then removed from the incubator and centrifugedat 300 g for ten minutes at 25° C. The medium is removed by tapping andblotting, and the cells are dried in a chemical fume hood for at leastfifteen minutes. 200 μl of fixative is added to every well and the plateis incubated for thirty minutes at room temperature. The cell fixativeis removed by tapping and blotting. 200 μl of blocking buffer are addedto every well, and the plate is incubated for thirty minutes at roomtemperature. The blocking buffer is removed by tapping and blotting, and100 μl of anti-BrdU working solution is added to each well and incubatedfor ninety minutes at room temperature. The anti-BrdU solution isremoved by tapping and blotting, and each well is rinsed three timeswith 300 μl washing solution. 100 μl 3.3′,5,5′-tetamethylbenzidine isadded to each well and incubated for ten minutes or until colorintensity is achieved. 25 μl 1M sulfuric acid is added to stop thereaction, and the plate is read on a fusion microplate reader(PerkinElmer) within five minutes. The results from the in vitroproliferation assay are presented in tables 1 and 2.

TABLE 1 Effect of DHA on Lymphocyte Proliferation PHA + PHA + 12.5 μM 25μM no PHA PHA DHA DHA Healthy cats 0.05 ± 0.05 0.32 ± 0.21 0.28 ± 0.190.25 ± 0.17 Cats with IBD 0.09 ± 0.11 0.58 ± 0.26 0.49 ± 0.15 0.45 ±0.18

TABLE 2 Effect of EPA on Lymphocyte Proliferation PHA + PHA + 12.5 μM 25μM no PHA PHA EPA EPA Healthy cats 0.05 ± 0.05 0.32 ± 0.21 0.38 ± 0.230.37 ± 0.23 Cats with IBD 0.09 ± 0.11 0.58 ± 0.26 0.62 ± 0.27 0.63 ±0.29

As can be seen from Tables 1 and 2, the proliferation activity of thelymphocytes obtained from the blood of the cats with IBD is higher thanthe proliferation activity of the lymphocytes obtained from the blood ofthe healthy cats. Incubation of lymphocytes from the cats with IBD withboth 12.5 and 25 μM DHA results in a decrease in lymphocyteproliferation. Incubation of lymphocytes from the cats with IBD withboth 12.5 and 25 μM EPA does not result in a decrease in lymphocyteproliferation.

Example 2

This example illustrates the effect of DHA and EPA on the cytokineprofile of lymphocytes obtained from the blood of healthy cats and catswith IBD.

Lymphocytes are obtained from the blood of healthy cats and cats withIBD as described in Example 1 and then treated with DHA or EPA also asdescribed in Example 1. Ribonucleic acid (RNA) is extracted from allsamples, and real time polymerase chain reaction (RT-PCR) is performedto examine the changes in the level of expression of the followingcytokines: interleukins 1α, 1β, 2, 6, and 10 (IL-1α, IL-1β, IL-2, IL-6,and IL-10), macrophage inhibitory factor (MIF), interferon gamma(IFN-γ), and transforming growth factor beta (TGF-β). The results fromthe cytokine PCR analysis are presented in Table 3.

TABLE 3 Effect of DHA and EPA on Cytokine Expression IL-1α IL-6 MIF IL-2IFN-γ IL-1β IL-10 TGF-β Healthy cats (no PHA) 1 1 1 1 1 1 1 1 Healthycats (PHA) 0.82 3.55 12.25 1.03 5.95 1.03 0.65 1.1 Healthy cats (PHA +DHA) 0.86 5.75 16.9 21.05 37.6 0.87 0.95 3.55 Healthy cats (PHA + EPA)1.05 10.6 14.05 59.4 130.9 1.17 1.2 4.7 Cats with IBD (no PHA) 0.355 *0.56 39.25 106 0.53 1.4 6.9 Cats with IBD (PHA) 0.57 5.1 10.45 7.9 23.750.63 0.37 6.95 Cats with IBD (PHA + DHA) 0.57 13.9 6.55 3 4.9 1.85 0.451.45 Cats with IBD (PHA + EPA) 0.46 6.45 4.45 1.89 4.45 1.34 0.34 2.4 *= undetected DHA = 12.5 μM DHA EPA = 12.5 μM EPA

Referring to Table 3, the level of expression of the proinflammatoryinterleukins IL-2 and IFN-γ is much higher in cats with IBD compared tohealthy cats, indicating the presence of inflammation. The level ofexpression of the anti-inflammatory cytokine TGF-β is slightly higher incats with IBD compared to healthy cats, probably to counteractinflammation. DHA decreases the level of expression of IL-2 and IFN-γ incats with IBD to a level similar to the level of expression of IL-2 andIFN-γ in the healthy cats stimulated with PHA in the absence of DHA. Thelevels of expression of cytokines in lymphocytes from cats with IBDtreated with PHA and DHA are similar to the levels of expression ofcytokines in lymphocytes from healthy cats treated with PHA in theabsence of DHA, suggesting that DHA normalizes the response of thelymphocytes from the cats with IBD to mitogen stimulation.

EPA also decreases the level of expression of IL-2 and IFN-γ in catswith IBD to a level similar to the level of expression of IL-2 and IFN-γin the healthy cats stimulated with PHA in the absence of EPA. Thelevels of expression of cytokines in lymphocytes from cats with IBDtreated with PHA and EPA are similar to the levels of expression ofcytokines in lymphocytes from healthy cats treated with PHA in theabsence of EPA, suggesting that EPA, like DHA, also normalizes theresponse of the lymphocytes from the cats with IBD to mitogenstimulation (although, as shown in Example 1, EPA does not decrease thelevel of lymphocyte proliferation in vitro).

In the specification, there have been disclosed typical preferredembodiments of the invention and, although specific terms are employed,they are used in a generic and descriptive sense only and not forpurposes of limitation, the scope of the invention being set forth inthe claims. Obviously many modifications and variations of the inventionare possible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims the inventionmay be practiced otherwise than as specifically described.

1-15. (canceled)
 16. A method for determining if a feline is sufferingfrom inflammatory bowel disease comprising: collecting lymphocytes fromblood obtained from the feline as a first sample; collecting lymphocytesfrom blood obtained from a healthy feline as a second sample; exposingthe second sample to an amount of a mitogen for a period of time;exposing the first sample to the same amount of the same mitogen as thesecond sample for the same period of time as the second sample in thepresence of an amount of docosahexaenoic acid or eicosapentaenoic acid;measuring the gene expression level of one or more of the cytokinesselected from the group consisting of interleukin-2 (IL-2) andinterferon gamma (IFN-γ); and comparing levels of gene expression in allsamples, wherein if the level of gene expression in the second sample issimilar to the level of gene expression in the first sample, then thefeline is suffering from inflammatory bowel disease.
 17. The method ofclaim 16 wherein the mitogen is selected from the group consisting ofphytohemagglutinin, concanavalin, pokeweed mitogen, lipopolysaccharide,and anti-CD3 antibody.
 18. (canceled)
 19. (canceled) 20-28. (canceled)